1. Field of the Invention
The present invention relates to the construction of six insertion (4, 8, 12, 18, 19 or 23 amino acid residues) and two deletion (4 or 7 amino acid residues) mutants of the linker region of FokI endonuclease from Flavobacterium okeanokoites. The mutant enzymes were purified, and their cleavage properties have been characterized.
2. Description of the Related Art
The FokI restriction endonuclease from Flavobacterium okeanokoites belongs to the Type IIS class of endonucleases. FokI recognizes the asymmetric sequence 5'-GGATG-3' and cleaves double-stranded DNA at staggered sites 9 and 13 nucleotides away from the recognition site (1, 2). The cloning and sequencing of the FokI restriction-modification system have been reported (3-4). Several research groups have purified FokI endonuclease and characterized its properties (5-9). Previous reports by the present inventor on proteolytic fragments of FokI endonuclease using trypsin have revealed a N-terminal DNA-binding domain and a C-terminal catalytic domain with non-specific DNA cleavage activity (10,11). These reports have suggested that the two domains are connected by a linker region which is susceptible to cleavage by trypsin. The present inventor has also shown that insertion of four (or seven codons) between the recognition and cleavage domains of FokI can alter the cleavage distance of FokI within its substrate (12).
Recently, Waugh and Sauer have shown that single amino acid substitutions uncouple the DNA-binding and strand scission activities of FokI endonuclease (13). Furthermore, they have obtained a novel class of FokI restriction mutants that cleave hemi-methylated DNA substrates (14). The modular structure of FokI suggested that it may be feasible to construct hybrid endonucleases with novel sequence-specificity by linking other DNA-binding proteins to the cleavage domain of FokI endonuclease. Recently, the present inventor reported the construction of the first "chimeric" restriction endonuclease by linking the Ubx homeo domain to the cleavage domain of FokI (15).
To further probe the linker region, the present inventor has constructed several insertion and deletion mutants of FokI endonuclease. A detailed description of the process for making and using and the properties of these mutants follows.